José A. Lemos

Recently, our group showed that the dental pathogen Streptococcus mutans is inherently more tolerant to high zinc stress than other streptococci, a phenotype associated with the presence of a P-type ATPase exporter named ZccE, virtually unique to S. mutans . In addition to zccE , a previous transcriptome analysis revealed that S. mutans upregulates genes involved in glutathione uptake during initial exposure to zinc stress. Glutathione, a major supplier of organic sulphur that also plays key roles in antioxidant defence and xenobiotic detoxification, forms coordination complexes with a variety of metals, including zinc, thereby functioning as a buffer that protects cells from metal intoxication. To investigate the contribution of glutathione zinc tolerance in S. mutans , the gshT gene, which encodes the substrate-binding subunit of a glutathione transporter, was deleted in both the parent and Δ zccE strains, and the ability of these mutants to overcome zinc stress through intracellular glutathione accumulation was determined. Targeted metabolomics revealed that S. mutans accumulates glutathione in a GshT-dependent manner following zinc stress, a response that was strikingly amplified in the Δ zccE strain. Although glutathione supplementation had a minimal impact on the growth of either parent or mutant strains in sub-inhibitory zinc concentrations, the Δ gshT strain exhibited increased zinc sensitivity in a plate-based assay. However, the Δ zccE Δ gshT mutant displayed enhanced zinc tolerance compared to the Δ zccE single mutant. While glutathione alone did not alter zinc levels in the UA159 or Δ zccE strains, the combination of zinc and glutathione nearly doubled intracellular zinc levels in Δ zccE compared to cells grown in zinc only. We conclude that while glutathione may play a minor role in S. mutans zinc tolerance, enhanced glutathione uptake observed in Δ zccE facilitates zinc entry, as glutathione:Zn ²⁺ complexes inadvertently promote zinc intoxication via a Trojan horse mechanism.

Recently, our group showed that the dental pathogen Streptococcus mutans is inherently more tolerant to high zinc stress than other streptococci, a phenotype associated with the presence of a P-type ATPase exporter named ZccE, virtually unique to S. mutans . In addition to zccE , a previous transcriptome analysis revealed that S. mutans upregulates genes involved in glutathione uptake during initial exposure to zinc stress. Glutathione, a major supplier of organic sulfur that also plays key roles in antioxidant defense and xenobiotic detoxification, forms coordination complexes with a variety of metals, including zinc, thereby functioning as a buffer that protects cells from metal intoxication. To investigate the contribution of glutathione zinc tolerance in S. mutans , the gshT gene, which encodes the substrate-binding subunit of a glutathione transporter, was deleted in both the parent and Δ zccE strains and the ability of these mutants to overcome zinc stress through intracellular glutathione accumulation determined. Targeted metabolomics revealed that S. mutans accumulates glutathione in a GshT-dependent manner following zinc stress, a response that was strikingly amplified in the Δ zccE strain. Although glutathione supplementation had a minimal and non-significant impact on growth of either parent or mutant strains in sub-inhibitory zinc concentrations, the Δ gshT strain exhibited increased zinc sensitivity in a plate-based assay. However, the Δ zccE Δ gshT mutant displayed enhanced zinc tolerance compared to the Δ zccE single mutant. While glutathione alone did not alter zinc levels in the UA159 or Δ zccE strains, the combination of zinc and glutathione nearly doubled intracellular zinc levels in Δ zccE compared to cells grown in zinc only. We conclude that while glutathione may play a minor role in S. mutans zinc tolerance, uncontrolled glutathione uptake observed in Δ zccE facilitates zinc entry, as glutathione:Zn ²⁺ complexes inadvertently promote zinc intoxication via a Trojan horse mechanism.

Enterococcus faecalis is a prolific opportunistic pathogen responsible for a range of life-threatening infections, notorious for its ability to withstand environmental stressors. Second messengers are small molecules that relay signals in response to stimuli and are thought to be crucial for bacteria like E. faecalis to modulate their adaptation to stress. The second messenger nucleotide c-di-AMP has emerged as an essential bacterial signaling molecule due to its impact on physiological processes, including adaptation to osmotic stress, cell wall homeostasis, antibiotic tolerance, and virulence. In addition, c-di-AMP is a pathogen-associated molecular pattern (PAMP) molecule that can trigger a potent stimulation of the host immune system. In previous work, we identified and characterized the enzymes responsible for the synthesis and degradation of intracellular c-di-AMP in E. faecalis , demonstrating that maintenance of c-di-AMP homeostasis is vital for its fitness and virulence. In addition to the intracellular enzymes that regulate c-di-AMP levels, a small number of bacteria encode surface-associated nucleotidases that cleave extracellular c-di-AMP and are potentially associated with immune evasion. Here, we characterize a novel and unique cell wall-anchored nucleotidase, termed EecP ( E . faecalis e xtracellular c -di-AMP p hosphodiesterase), which features duplicated catalytic domains and degrades extracellular c-di-AMP. Through competition experiments, we show that EecP likely uses c-di-GMP, and to a lesser extent AMP, as additional substrates. While a Δ eecP strain failed to display relevant phenotypes under most in vitro conditions, it exhibited increased susceptibility to killing by phagocytic cells, a phenotype at least partly associated with cGAS-STING immune signaling. NanoString analysis revealed distinct innate immune signatures in murine macrophages infected with the parent strain OG1RF or Δ eecP , uncovering differential expression of host targets known to be impacted by c-di-AMP, as well as novel targets. Using two murine infection models, we show that the impact of eecP deletion and the consequent buildup of extracellular c-di-AMP on E. faecalis pathogenesis might depend on the site of infection. Notably, disseminated infection was more severe in mice infected with Δ eecP, suggesting that extracellular c-di-AMP influences infection outcomes, likely through modulation of host immune responses.

From its discovery in the late 1800s to the present day, Streptococcus mutans research has been at the forefront of dental caries investigations, providing the foundation for our current understanding of the microbiological determinants of this disease. In addition, research on S. mutans has greatly advanced key areas of microbiology, establishing the organism as a notably useful model for understanding the biology of other streptococcal species, as well as Gram-positive bacteria more broadly. In this piece, we provide a retrospective of how S. mutans has served this dual role, highlighting specific areas that have benefited most from S. mutans –driven discoveries. We close by outlining emerging areas of S. mutans research that are likely to influence future breakthroughs in both oral health science and basic microbiology.

Halogenated phenazine (HP) compounds have shown promise as antimicrobial agents, particularly against biofilm-associated Gram-positive pathogens. Among these compounds, HP-29 demonstrates potent activity against methicillin-resistant Staphylococcus aureus by inducing rapid iron starvation. As maintenance of trace metals homeostasis is critical for the survival of Streptococcus mutans , this study investigated the antimicrobial efficacy of HP-29 and the impact of metal supplementation on this major oral and occasional systemic pathogen. As anticipated, HP-29 inhibited S. mutans growth in a dose-dependent manner, with iron supplementation alleviating the antimicrobial effect. Cobalt, manganese, or nickel supplementation also mitigated the inhibitory activity of HP-29, but, unexpectedly, the addition of zinc greatly enhanced HP-29 antimicrobial activity. This zinc-driven potentiation of HP-29 extended to other Gram-positive pathogens, including Enterococcus faecalis and S. aureus . Inductively coupled plasma mass spectrometry analysis revealed that intracellular iron content decreased significantly following exposure to HP-29. When combined with zinc, HP-29 triggered a 5-fold increase in intracellular zinc and reduced manganese levels by ~50%. Transcriptome analysis showed that HP-29 treatment, with or without zinc, altered expression of genes linked to iron and manganese uptake as well as zinc efflux, suggesting broad disruption of metal ion regulation. These findings highlight HP-29 as a potent antimicrobial that broadly impairs metal homeostasis. The unexpected synergy of HP-29 with zinc points toward a promising dual-agent therapeutic strategy against Gram-positive pathogens. IMPORTANCE: Widespread development of antibiotic resistance has created a constantly moving target when combating infectious microbes. Here, we further explore an antimicrobial halogenated phenazine, HP-29, which is effective against Gram-positive bacteria through disruption of intracellular trace metal equilibrium. We showed that HP-29 inhibits growth of the oral and systemic pathogen Streptococcus mutans and that its antimicrobial effect is greatly potentiated by the addition of zinc. The zinc-mediated enhancement of HP-29’s efficacy was also observed in other Gram-positive pathogens, including Enterococcus faecalis and Staphylococcus aureus . Intracellular trace metal quantifications and transcriptome analysis confirmed that HP-29 treatment impairs trace metal homeostasis, an outcome that is exacerbated when S. mutans is treated with both HP-29 and zinc. The observed synergy of HP-29 with zinc supports the development of a dual-agent therapeutic strategy against Gram-positive pathogens.

Wound infections remain an important medical problem, which is aggravated by the prevalence of multidrug-resistant bacteria. Among them, Enterococcus faecalis is a major pathogen of surgical site incisional and diabetic chronic wounds, but factors driving its colonization and persistence in wounds remain poorly understood. Iron, manganese, and zinc are essential cofactors in cellular processes, prompting the host to restrict their availability through mobilization of metal-sequestering proteins, a defense known as nutritional immunity. Previously, we showed that E. faecalis strains lacking key iron (Δ5Fe), manganese (Δ3Mn), or zinc (Δ2Zn) uptake systems have impaired virulence. Here, we used an excisional wound model in normoglycemic (C57Bl/6J or B6) and diabetic (C57Bl/6J lepR ^−/− or DB) mice to examine the role of these metal import systems in wounds. The strong upregulation of metal import genes and reduced wound colonization by Δ3Mn, Δ5Fe, and Δ2Zn strains in B6 mice indicate that iron, manganese, and zinc are limited during wound infection. While Δ2Zn and Δ3Mn strains showed no improved colonization in diabetic wounds, the Δ5Fe strain exhibited a temporary colonization advantage over non-diabetic mice. Quantifications of metal-sequestering proteins lactoferrin, transferrin, calprotectin, and psoriasin from intact skin and infected wounds indicated that nutritional immunity, especially iron restriction, is delayed in diabetes. In conclusion, this study underscores the crucial role of trace metal acquisition in E. faecalis wound colonization and suggests differences in metal bioavailability between diabetic and non-diabetic wounds, helping to explain the increased susceptibility of diabetic wounds to chronic infection.

Wound infections remain an important medical problem, which is aggravated by the prevalence of multidrug resistant bacteria. Among them, Enterococcus faecalis is a major pathogen of surgical site incisional and of diabetic chronic wounds, but factors driving its colonization and persistence in wounds remain poorly understood. Iron, manganese, and zinc are essential cofactors in cellular processes, prompting the host to restrict their availability through mobilization of metal-sequestering proteins, a defense known as nutritional immunity. Previously, we showed that E. faecalis strains lacking key iron (Δ5Fe), manganese (Δ3Mn), or zinc (Δ2Zn) uptake systems have impaired virulence. Here, we used an excisional wound model in normoglycemic (C57Bl/6J or B6) and diabetic (C57Bl/6J lepR⁻/⁻ or DB) mice to examine the role of these metal import systems in wounds. The strong upregulation of metal import genes and reduced wound colonization by Δ3Mn, Δ5Fe, and Δ2Zn strains in B6 mice indicate that iron, manganese, and zinc are limited during wound infection. While Δ2Zn and Δ3Mn strains showed no improved colonization in diabetic wounds, the Δ5Fe exhibited a temporary colonization advantage over non-diabetic mice. Quantifications of metal-sequestering proteins lactoferrin, transferrin, calprotectin and psoriasin from intact skin and infected wounds indicated that nutritional immunity, especially iron restriction, is delayed in diabetes. In conclusion, this study underscores the crucial role of trace metal acquisition in E. faecalis wound colonization and suggests differences in metal bioavailability between diabetic and non-diabetic wounds, helping to explain the increased susceptibility of diabetic wounds to chronic infection.

The collagen‐binding adhesin Cnm is a known virulence factor of Streptococcus mutans . It is present in specific serotypes (mostly e, f, and k strains) of S. mutans and belongs to the LPXTG family of cell wall‐anchored surface adhesins. Here, we report the crystal structure of the collagen‐binding N ₂ domain of S. mutans Cnm. Using the Staphylococcus aureus collagen‐binding protein Cna, which shares high sequence and structural homology with Cnm, we modeled collagen binding to S. mutans Cnm. The comparative analysis identified three conserved collagen‐binding residues (Y176, F192, N194) and four equivalent residues that are different in their composition (D224, T226, S232, M276). This study also discovered the multifunctional attributes of this protein, where Cnm‐FL, Cnm‐N _12, and the individual domains of Cnm‐N ₁ and Cnm‐N ₂ adhere with high affinity to the scavenger receptor cysteine‐rich (SRCR) domains of glycoprotein 340 (Gp340). Protein–protein docking of Cnm‐N ₂ and SRCR ₁ showed the possibility of a shared binding site at the collagen‐binding interface of Cnm‐N ₂ . Furthermore, competition experiments using collagen and SRCR ₁₂₃ with Cnm‐N ₂ , Cnm‐N _12, and Cnm‐FL constructs confirmed that collagen and SRCR ₁ share a binding site. Subsequent alanine substitution mutagenesis of the predicted collagen‐binding residues validated our modeling results, confirming that Y176 and F192 are important residues for collagen and SRCR/Gp340 binding.

Halogenated phenazine (HP) compounds have shown promise as antimicrobial agents, particularly against biofilm-associated Gram-positive pathogens. Among these compounds, HP-29 demonstrates potent activity against methicillin-resistant Staphylococcus aureus by inducing rapid iron starvation. As maintenance of trace metals homeostasis is critical for the survival of Streptococcus mutans , this study investigated the antimicrobial efficacy of HP-29 and the impact of metal supplementation on this major oral and occasional systemic pathogen. As anticipated, HP-29 inhibited S. mutans growth in a dose-dependent manner, with iron supplementation alleviating the antimicrobial effect. Cobalt, manganese, or nickel supplementation also mitigated the inhibitory activity of HP-29 but, unexpectedly, the addition of zinc greatly enhanced HP-29 antimicrobial activity. This zinc-driven potentiation of HP-29 extended to other Gram-positive pathogens, including Enterococcus faecalis and S. aureus . Inductively coupled plasma mass spectrometry analysis revealed that intracellular iron content decreased significantly following exposure to HP-29. At the same time, exposure to HP-29 led to a slight increase in intracellular zinc, mirroring the increase observed in cells exposed to excess zinc. When combined with zinc, HP-29 triggered a 5-fold increase in intracellular zinc and reduced manganese levels by ∼50%. Transcriptome analysis showed that HP-29, with or without zinc, altered expression of genes linked to iron and manganese uptake as well as zinc efflux, suggesting broad disruption of metal ion regulation. These findings highlight HP-29 as a potent antimicrobial that broadly impairs metal homeostasis. The unexpected synergy of HP-29 with zinc points toward a promising dual-agent therapeutic strategy against Gram-positive pathogens. IMPORTANCE: Widespread development of antibiotic resistance has created a constantly moving target when combating infectious microbes. Here, we further explore an antimicrobial halogenated phenazine, HP-29, which is effective against Gram-positive bacteria through disruption of intracellular trace metal equilibrium. We showed that HP-29 inhibits growth of the oral and systemic pathogen Streptococcus mutans and that its antimicrobial effect is greatly potentiated by the addition of zinc. The zinc-mediated enhancement of HP-29’s efficacy was also observed in other Gram-positive pathogens, including Enterococcus faecalis and Staphylococcus aureus . Intracellular trace metal quantifications and transcriptome analysis confirmed that HP-29 treatment impairs trace metal homeostasis, an outcome that is exacerbated when S. mutans is treated with both HP-29 and zinc. The observed synergy of HP-29 with zinc supports the development of a dual-agent therapeutic strategy against Gram-positive pathogens.